acase@tulane.edu / Osteoporosis is a prevalent bone metabolic disease characterized by bone fragility. As a key pathophysiological mechanism, the disease is caused by excessive bone resorption (by osteoclasts) over bone formation (by osteoblasts). Peripheral blood monocytes (PBMs) represent a major systemic cell type for bone metabolism by serving as progenitors of osteoclasts and producing cytokines important for osteoclastogenesis. Our lab previously used microarray-based transcriptomics profiling to identify a list of novel genes for osteoporosis. My work is to further investigate the factors and regulatory network in osteoporosis, using microarray data of monocytes from subjects with extremely high/low hip bone mineral density. 1) We performed a pathway analysis and developed a novel approach to correct the “crosstalk” phenomenon which is caused by overlapping genes. 2) We analyzed the long non-coding RNA (lncRNA) profile by re-annotating exon array and predicted the regulatory mechanism of lncRNAs on protein coding genes in bone metabolism. 3) We identified the important potential transcription factors for osteoporosis and inferred the regulatory mechanism which exists between transcription factors and target genes in bone metabolism. My findings not only reported the key regulatory factors (lncRNAs and transcriptional factors) contributes to bone metabolism, but also explored the potential regulatory networks in osteoporosis. / 1 / Yu Zhou

No description available.

Au sein des hémopathies malignes B, les plasmocytoses malignes (myélome multiple [MM) et leucémie à plasmocyte [PCLI) occupent une place particulière par leur biologie et leurs aspects cliniques. Biologiquement, elles présentent une forte proportion d’anomalies oncogéniques· (RAS, c-MYC) et de fréquentes altérations de la voie p53 (CDKN2ADel, TP53Del/Mut) qui conduisent, cliniquement, à l’inefficacité des traitements cytostatiques conventionnels. Des lignées cellulaires de MM (HMCls) qui recouvrent en partie la diversité des patients ont été générées depuis 50 ans. Nous avons caractérisé l’exome complet de 33 lignées cellulaires humaines de MM. Les mutations faux-sens sont les plus fréquentes (92%). les HMCLs portent entre 307 et 916 mutant par HMCL, TP53 étant le gène le plus altéré (67%). Des pertes bi-alléliques des voies du cycle cellulaire (CDKN2C, RB1), de la voie NFkB (TRAF3, BIRC2) et de la voie p53 (TP53, CDKN2A) sont fréquentes. La fréquence des mutations/délétion est semblable à celle des patients ( DIS3, PRDM1, KRAS), ou majorée (TP53, CDKN2C, NRAS, PRKD2). la voie MAPK est lá plus altérée (82% des HMCls), principalement par des mutants de RAS: peu décrites, les HMCLs présentent des altérations des voies épigénétiques (73%), de l’ anémie de Fanconi (54%) et très peu d’anonalies directes de la machinerie apoptotique. Nous avons mis en relation les données dexpression, de mutation/délétion et de réponse aux traitements et démontré que l’efficacité de plusieurs traitements est indépendante des mutations. Finalement, le développement de stratégies prenant en compte ces altérations peu décrites dans le MM (Fanconi, Epigenetique) sont nécessaires. / Among B CeH malignancies, plasma-cell the NFKB pathway (TRAF3, BIRC2) and the p53 malignancies (multiple myeloma [MM] and plasma cell pathway (TP53, CDKN2A). Frequency of leukemia [PCL]) harbor particular biological and mutations/deletions in HMCLs were either similar to clinical insights. Biologically, they present with both a that of patients (e.g. DIS3, PRDM1, KRAS), or highly high frequency of oncogenic abnormalities (RAS, e- increased (e.g. TP53, CDKN2C, NRAS, PRKD2). MYC) and a high frequency of p53 pathway MAPK was the most altered pathway (82% of abnormalities (CDKN2Adel, TP53 del/mut). Those two HMCLs), mainly by RAS mutants. Surprisingly, latter leading to chemo-resistance to conventional HMCLs displayed alterations in epigenetic (73%) and cytostatic drugs. Human myeloma cell lines (HMCLs) Fanconi anemia (54%) and few alterations in are widely used for their representation of primary apoptotic machinery. We further identified mutually myeloma cells as they cover patient diversity, exclusive and associated mutations/deletions in afthough not fully. We performed whole-exon genes involved in the MAPK and p53 pathways as sequencing of 33 HMCLs, which were established well as in chromatin regulator/modifier genes. over the last 50 years. Missense mutations were the Finally, by combining the gene expression profile, most frequent mutations {92%). HMCLs harbored gene mutation. gene deletion and drug response, we between 307 and 916 mutations per sample, with demonstrated that several targeted drugs overcome TP53 being the most mutated gene (67%). Recurrent or bypass some mutations bi-allelic losses were found in genes involved in cell cycle regulation (RB1. CDKN2C).

No description available.

There are numerous different white spotting patterns in the horse, including two of particular interest tobiano and sabino. In the mouse, genetic variation in the gene KIT causes many white spotting patterns. Due to the phenotypic similarity among white spotting patterns in horses and mice, KIT was investigated as the cause of the tobiano and sabino spotting patterns in horses. Initially, the KIT cDNA sequences from horses with several spotting patterns were compared. Three single nucleotide polymorphisms (SNPs) were identified, though none were associated with a spotting pattern. Three novel splicing variants were also observed: exon 17 skipping, exon 18 skipping and alternative splicing of exon 3. Families segregating for a sabino spotting pattern (designated Sabino 1) and exon 17 skipping were discovered. Sequencing revealed a SNP (KI16+1037) within intron 16 that was completely associated with skipping of exon 17. Using a PCR-RFLP for KI16+1037, linkage was discovered for sabino spotting (LOD=9.02 for =0) and presence of the Sabino 1 allele detected in seven breeds. While all horses with this SNP exhibited the Sabino 1 phenotype, some horses with a sabino phenotype did not possess the SNP. This is most likely due to genetic heterogeneity of the phenotype. Fluorescent in situ hybridization (FISH) was used to investigate the possibility of chromosome inversion in the region of KIT. A chromosomal inversion was discovered spanning ECA3q13 to 3q21 using BAC clones containing KIT and other genes in the same region. The ECA3q inversion was completely associated with Tobiano in the eight horses tested by FISH. This inversion may disrupt regulatory sequences of the KIT gene and thereby cause tobiano spotting. Spotting patterns are important to horse breeders for aesthetic as well as economic reasons. Spotting patterns in the horse may also be an interesting scientific model. The two genetic variants discovered in this work are good examples of genetic diversity due to mechanisms other than SNPs. Study of these variants may be valuable for examining the effects of the KIT gene on health traits. In particular, the KIT gene directs many functions of the mast cell, a cell that is involved in the etiology of inflammation.

Made available in DSpace on 2016-06-02T20:21:36Z (GMT). No. of bitstreams: 1 6342.pdf: 2523813 bytes, checksum: 92295d7cb18d3fbd0dcbfe0d0cbbcf45 (MD5) Previous issue date: 2014-09-05 / Universidade Federal de Minas Gerais / Schistosoma mansoni is the causative agent of Schistosomiasis, reaching 249 million people in 78 countries. It is believed that this infection is successful due to modulation of the host immune system through proteins secreted by the parasite. Recently a class of proteins derived from micro-exon genes (MEGs) was observed in proteomic analyses of schistosomula and egg secretions of S. mansoni, and also associated with glands of several stages of the parasite life. Furthermore, they found that none of these proteins showed similarity to proteins of organisms from other genres. In this context, aim of this work was the structural study, the search for protein partners and biological assays seeking possible roles for the proteins encoded by genes of micro-exons of S. mansoni: MEG-5, MEG-8.2 e MEG-12. For structural characterization studies we used MEG-5 protein expressed by heterologous system (the expression of MEG-8.2 protein was not obtained) and initially performed tests Circular Dichroism (CD). This essay revealed the predominance of one disordered structure due to the presence of a negative peak in the ranges of 180-200mm and the presence of lower intensity peaks in the range 220- 240mm, indicating that part of the protein may present structure. Such structure was observed in the secondary structure predictions performed in SOPMA and Jpred3 programs, wherein the presence of a predicted alpha-helix in the C-terminal region. To confirm this fact, we performed an analysis of the maximum emission of tryptophan fluorescence by the technique of intrinsic fluorescence, because the protein has a single tryptophan present in the probable alpha-helical region. We note that the maximum fluorescence emission is at 330mm, indicating that it is in more hydrophobic environment. After the structural characterization of MEG-5 protein we seek partners through the assay two-hybrid system performing a scan on leukocyte cDNA libraries, yielding two possible partners. The CHMP1B protein was the one that drew the most attention in for being part of the formation of vesicular body and be related to the processes of antigen presentation. The synthetic peptide MEG-12 has a peculiar characteristic of an amphipathic helix predicted by Jpred3 program, and hemolytic character. This action was best observed by scanning electron microscopy (SEM), to be verifies in a clear disruption of the membrane to the breakup. The peptide is secreted into the esophagus may region associated with the digestion of erythrocytes made by the parasite. Therefore, these two proteins are shown to be important in the host-parasite relationship and possible targets for drugs or creation of more efficient vaccines. / O Schistosoma mansoni é o agente causador da esquistossomose mansônica, atingindo 249 milhões de pessoas em 78 países. Acredita-se que tal infecção é bem-sucedida devido a modulação do sistema imune do hospedeiro por meio de proteínas secretadas pelo parasita. Recentemente uma classe de proteínas derivadas de genes de micro-exon (MEGs) foi observada em análises proteômicas de secreções de esquistossômulo e ovo, e também associadas com glândulas de vários estágios de vida do parasita. Além disso, verificou que nenhuma destas proteínas apresentavam similaridade com proteínas de organismos de outros gêneros. Neste contexto, objetivo deste trabalho foi o estudo estrutural, a busca por parceiros protéicos e ensaios biológicos buscando possíveis funções para as proteínas codificadas por genes de micro-exons de S. mansoni: MEG-5, MEG-8.2 e MEG-12. Para os estudos de caracterização estrutural utilizamos a proteína MEG-5 expressa pelo sistema heterólogo (não obtivemos a expressão suficiente da proteína MEG-8.2) e inicialmente realizamos os ensaios de Dicroísmo Circular (CD). Este ensaio nos revelou a predominância de uma estrutura desordenada, devido a presença de um pico negativo nos intervalos de 180-200nm e a presença de picos de menor intensidade na faixa entre 220-240nm, indicando que parte da proteína pode apresentar estruturação. Tal estruturação foi observada nas predições de estrutura secundária realizadas nos programas SOPMA e Jpred3, em que, previram a presença de uma alfa-hélice na região C-terminal. Para a confirmação deste fato, realizamos uma análise do máximo de emissão de fluorescência no triptofano pela técnica de fluorescência intrínseca, pois a proteína possui um único triptofano presente na região da provável alfa-hélice. Constatamos que o máximo de emissão de fluorescência está em 330nm, indicando que este se encontra em ambiente mais hidrofóbico. Após a caracterização estrutural da MEG-5 buscamos parceiros protéicos por meio do ensaio de dublo hibrido realizando uma varredura em bibliotecas de cDNA de leucócitos, obtendo dois possíveis parceiros. A proteína CHMP1B foi a que mais nos chamou atenção, por fazer parte da formação do corpo vesicular e estar relacionada aos processos de apresentação de antígenos. O peptídeo codificado por MEG-12 apresenta uma característica peculiar de uma hélice anfipática, predita pelo programa Jpred3, e um caráter hemolítico. Sua ação em eritrócitos foi caracterizada por microscopia eletrônica de varredura (MEV), que verificou mudanças morfológicas das células, sugerindo a perturbação na membrana até o rompimento. O peptídeo é secretado na região do esôfago podendo está relacionado com a digestão de eritrócitos realizada pelo parasita. Por tanto, esta duas proteínas demonstram ser importantes na relação parasitohospedeiro e possíveis alvos para criação de medicamentos ou vacinas mais eficientes.

La protéine MLN51, surexprimée dans environ 30% des cancers du sein, est un facteur clé du métabolisme des ARNm, en tant que membre du complexe de jonction des exons (EJC). Ce graffiti moléculaire est un régulateur essentiel de l’expression génique de par son implication dans l’épissage, l’export, la traduction, la stabilité et la dégradation des ARNm. A l’échelle structurale, l’EJC est organisé autour d’un coeur protéique avec l’hélicase eIF4A3, MLN51 et l’hétérodimère Magoh/Y14. Ce tétramère sert de plateforme d’ancrage à d’autres facteurs périphériques. Les mécanismes de recrutement du coeur EJC sur l’ARNm ont été élucidés par des approches biochimiques. Dans ce contexte, nous avons initié un travail original destiné à mettre en évidence la localisation cellulaire de l’assemblage du coeur EJC in vivo. L’utilisation de techniques d’imagerie photonique et électronique a permis d’établir un lien véritable entre la localisation du coeur EJC et l’architecture nucléaire. Nous avons montré que la plupart des facteurs EJC sont localisés et interagissent à la périphérie des speckles nucléaires, lieux de stockage des facteurs d’épissage. Ces régions discrètes nucléaires ont été appelées «perispeckles» et sont des entités distinctes des speckles. De manière intéressante, la localisation des protéines coeur coïncide avec celle des ARNm dans les perispeckles et est spatialement reliée aux sites transcriptionnels. Ces données démontrent que l’assemblage du coeur EJC a lieu dans le compartiment nucléaire et définissent le perispeckle comme un territoire intermédiaire entre les speckles nucléaires et sites de transcription où s’opèrent des évènements post-transcriptionnels fondamentaux. / The MLN51 protein, overexpressed in around 30% of breast cancers, is a key factor for mRNA metabolism, as a member of the Exon Junction Complex (EJC). The EJC marks the splicing history of an mRNA and influences many stages of its subsequent metabolism: splicing, dynamic cytoplasmic export, efficient and localized translation, quality-control and stability. Structurally, the EJC is organized around a core complex that is formed by four proteins (eIF4A3, MLN51, Magoh, Y14). The core complex serves as a binding platform for more than a dozen peripheral factors. The EJC is not a pre-assembled complex; however, its assembly mode is well described in vitro using recombinant proteins and splicing extracts. Nevertheless, where this complex assembles in vivo was a matter of debate. By using light and electron microscopy approaches, we established an original link between the cellular distribution of the EJC core factors and the nuclear architecture. The core and most of the peripheral EJC factors are colocalized and interact together in discrete regions of the nucleus, located at the periphery of nuclear speckles. This doughnut-shaped region appears to be a novel nuclear territory that we termed “the perispeckle”. This territory is distinct from nuclear speckles; it contains nascent mRNAs and it is close to active transcription sites. Overall, this study supports a model in which the deposition of the EJC core takes place in the nucleus, and that assembled EJC core factors concentrate in discrete subnuclear territories termed perispeckles. These regions contribute to the compartmentalization of the nucleus as an active domain implicated in mRNP packaging.

<p>HIV-1 relies on both viral and cellular host factors for expression of its genome. Tat specific factor 1 (Tat-SF1) was identified as a cellular cofactor required for enhanced transcription of HIV-1 <italic>in vitro</italic>. Insight into the role of Tat-SF1 in the HIV-1 lifecycle has previously been limited to immunodepletions and <italic>in vitro</italic> analyses or transient overexpression experiments. Here, we present studies that utilize RNA interference (RNAi) to reevaluate Tat-SF1's role in Tat transactivation and HIV-1 replication <italic>in vivo</italic>. We report that although Tat-SF1 depletion reduces HIV-1 infectivity, it does not affect Tat transactivation <italic>in vivo</italic>. However, Tat-SF1 depletion changes the levels of unspliced and spliced RNAs. We propose that Tat-SF1 has a novel role of post-transcriptionally regulating HIV-1 gene expression, possibly through alternative splicing.</p><p>The functions of Tat-SF1 in cellular gene expression are not well understood, so we utilized the stable cell lines constructed for our HIV-1 studies to investigate the cellular functions of Tat-SF1. To identify target genes of Tat-SF1, we employed a combination of RNAi and human exon arrays. These arrays, which survey both transcript-level and exon-level changes genome-wide, revealed approximately 1,400 genes with alternative exon usage after Tat-SF1 depletion (p&le;0.01). In contrast, 500 genes showed significant transcript-level changes (p&le;0.01), all with minimal fold changes. Computational analyses showed that genes with alternative exon usage after Tat-SF1 depletion were over-represented in the insulin signaling and ubiquitin mediated proteolysis biological pathways. Furthermore, there was approximately 2-fold enrichment of Tat-SF1 target genes among previously reported HIV-1 dependency factors. The type of exon choice affected by Tat-SF1 depletion exhibited a strong 5&rsquo; bias. Finally, a novel Tat-SF1 binding motif, GACGGG, was found to be over-represented among target genes and may play a functional role in first exon choice. Together, these data are the strongest evidence to date of Tat-SF1 functioning in both transcription and splicing of cellular genes.</p> / Dissertation

beta-Bungarotoxin (beta-Bgt), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, two novel beta-Bgt isotoxins were purified from Bungarus multicinctus venom by the combinations of ion-exchange chromatography and reverse phase HPLC. Amino acid sequencing, peptide mapping and mass analyses revealed that they probably contained the same A chain, but their B chain differed. Consequently, the discrepancies in their biological activity and fine structure reflected the role of B chain in intact of beta-Bgt. In Yeast-Two-Hybrid system, a potassium channel binding protein was identified to interact with the B chain of beta-Bgt. Although the recombinant potassium channel binding protein functionally bound with Ca2+, but it could not prove to bind with BM12 and BM13 as revealed by in vitro cross-linking assay. The A chain genes including A1 chain, A2 chain and A8 chain genes were amplified by PCR reaction. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with A chain cDNAs revealed that the A1 chain gene was organized with four exon and three intron, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponds to the first exon of A1 chain gene is skipped instead of inclusion of intronic sequence adjacent to the second intron. The resulting A2 chain mRNA encoded a 25 residues signal peptide, which different from A1 chain mRNA with a 27 residues signal peptide. Comparative analyses on phospholipase A2 genes and cDNAs suggest that this is the first report on skipping of exon changes the signal peptide sequence of snake venom proteins.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21664号 / 医博第4470号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 浅野 雅秀, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM